Differential expression of guinea pig class II major histocompatibility complex antigens on vascular endothelial cells in vitro and in experimental allergic encephalomyelitis

Previous studies have shown that vascular endothelial cells do not normally express major histocompatibility complex (MHC) Class II antigens either in vivo or in vitro. In this investigation it was found that endothelial in the central nervous system (CNS) of normal guinea pigs constitutively express MHC Class II antigens recognized by the monoclonal antibodies HLA-DR, 27E7, and MSgp8. This phenotype is retained when these CNS-derived endothelial cells are propagated in tissue culture. Furthermore, examination of CNS tissue taken from animals in the acute phase of chronic relapsing experimental allergic encephalomyelitis shows that additional epitopes of the MHC Class II antigen, detected by the monoclonal antibodies CI.13.1 and 22C4, are present during the diseased state. This study not only demonstrates constitutive expression of certain MHC Class II determinants by guinea pig endothelial cells, but also shows that other Class II determinants can be differentially expressed in certain disease states.     

Chemical synthesis of a gene for human stefin A and its expression in E. coli

A DNA containing the coding sequence for the human cysteine proteinase inhibitor stefin A was obtained by enzymic ligation of chemically synthesized deoxyoligonucleotides, using the Khorana ligation method. The 306-bp synthetic gene carries signals for the initiation and termination of its translation. The gene was expressed in E. coli using a cytoplasmic expression vector and stefin A was secreted under the control of the E. coli alkaline phosphatase signal sequence, respectively. The secreted hybrid protein was shown to exhibit biological properties similar to the native protein isolated from human plasma.     

Effect of anticancer drugs on the release of interleukin-6 in vitro

Summary This study investigates the effects of anticancer drugs and immunomodulating agents on the release of interleukin-6 (IL-6) from lipopolysaccharide-stimulated human peripheral blood mononuclear leucocytes in vitro. The addition of non-cytotoxic concentrations of Adriamycin (doxorubicin), vincristine and 4-OOH-cyclophosphamide (the in vitro active analogue of cyclophosphamide) resulted in suppression of IL-6 release. The drugs bleomycin, FK156 [d-lactoyl-l-alanyl--d-glutamyl-(l)-meso-diaminopimelyl-(l)-glycine], FK565 [heptanoyl--d-glutamyl-(l)-meso-diaminopimelyl-(d)-alanine] and the immunosuppressive agent cyclosporin A did not alter the release of IL-6 in the same experimental system.     

Structural differences between rabbit cathepsin E and cathepsin D

Rabbit cathepsins D and E were isolated from bone marrow. Both enzymes were purified by affinity chromatography on pepstatin-Sepharose 4B and Con A-Sepharose 4B. Purity of the enzymes was ascertained by two-dimensional gel electrophoresis after iodination. The isoelectric point of cathepsin D was found to be 6.95. Cathepsin E was shown to consist of two subunits having molecular masses each of 40 kDa and isoelectric points of 4.60 and 4.65, respectively. The amino-acid composition of cathepsin E was found to be different from that of cathepsin D.     

Streptomyces rimosus extracellular proteases 3. Isolation and characterization of leucine aminopeptidase

Summary A leucine aminopeptidase was purified to homogeneity fromStreptomyces rimosus culture filtrates, which are waste broth of oxytetracycline bioproduction process. Purification procedure includes ultrafiltration and chromatography on CM-Sephadex, AH-Sepharose and FPLC Mono S column. The enzyme is a monomer with molecular weight of 27,500 Daltons and pI of 7.3, stable in broad pH range and up to 70°C. It is a metallo enzyme dependent on Ca²⁺ ions for its full activity. By its specificity it is a true aminopeptidase active on amino acid amide, arylamide, peptide and ester bonds. The hydrolysing activity shows preference for leucine at the N-terminal position of substrates, also acts on aromatic acids and methionine, but does not release glycine, proline, acidic amino acids orD-amino acid residues.     

Effect of anticancer drugs on the release of interferon γ in vitro

Summary Some conventional and experimental anticancer drugs were tested for their effect on concanavalin-A-induced interferon release from rat splenocytes in vitro. When 2.5 × 10⁶ rat splenocytes/ml, stimulated with 1 µg/ml concanavalin A, were incubated with various non-cytotoxic doses of the vinca alkaloid vincristine, there was an inhibition of the release of interferon in culture supernatants. The antitumour antibiotics bleomycin and Adriamycin, alkylating agents 4-hydroperoxycyclophosphamide and mafosfamide, and the immunoactive peptides FK 156 and FK565 did not affect the release of interferon under similar conditions. However, cyclosporin A, in similar experiments, markedly inhibited the release of interferon .     

Mutations which affect the inhibition of protein phosphatase 2A by simian virus 40 small-t antigen in vitro decrease viral transformation.

Three independent point mutations within residues 97 to 103 of the simian virus 40-small-t antigen (small-t) greatly reduced the ability of purified small-t to inhibit protein phosphatase 2A in vitro. These mutations affected the interaction of small-t antigen with the protein phosphatase 2A A subunit translated in vitro, and a peptide from the region identified by these mutations released the A subunit from immune complexes. When introduced into virus, the mutations eliminated the ability of small-t to enhance viral transformation of growth-arrested rat F111 cells. In contrast, the mutant small-t antigens were unimpaired in the transactivation of the adenovirus E2 promoter, an activity which was reduced by a double mutation in small-t residues 43 and 45.     

Platelet activating factor synthesis and metabolism in intestinal ischaemia-reperfusion injury

The object of this study was to characterize the synthesis and metabolism of platelet activating factor (PAF) by intestinal mucosa subjected to ischaemia-reperfusion injury. Canine intestinal mucosa produced 16:0-PAF, 18:0-PAF, and high levels of the corresponding lyso- PAF metabolites. Three h of intestinal ischaemia and ischaemia followed by 1 h of reperfusion did not affect the synthesis or metabolism of PAF by intestinal mucosa. Intestinal mucosa elaborated a factor that rapidly hydrolyzes PAF to lyso-PAF. The observed hydrolysis rate was not altered by ischaemia or ischaemia and reperfusion. In conclusion, this study suggests that intestinal mucosa produces PAF and rapidly hydrolyzes PAF. The PAF synthesis and metabolism rates of intestinal mucosa is not altered by ischaemia reperfusion in this model under the imposed conditions.